Macaque monkey trigeminal blink reflex circuits targeting orbicularis oculi motoneurons.

The trigeminal blink reflex plays an important role in protecting the corneal surface from damage and preserving visual function in an unpredictable environment. The closing phase of the human reflex, produced by activation of the orbicularis oculi (ObOc) muscles, consists of an initial, small, ipsilateral R1 component, followed by a larger, bilateral R2 component. We investigated the circuitry that underlies this reflex in macaque (Macaca fascicularis and Macaca mulatta) monkeys by the use of single and dual tracer methods. Injection of retrograde tracer into the facial nucleus labeled neurons in the principal trigeminal nucleus, and in the spinal nucleus pars oralis and interpolaris, bilaterally, and in pars caudalis, ipsilaterally. Injection of anterograde tracer into the principal trigeminal nucleus labeled axons that directly terminated on ObOc motoneurons, with an ipsilateral predominance. Injection of anterograde tracer into pars caudalis of the spinal trigeminal nucleus labeled axons that directly terminated on ipsilateral ObOc motoneurons. The observed pattern of labeling indicates that the reticular formation ventromedial to the principal and spinal nuclei also contributes extensive bilateral input to ObOc motoneurons. Thus, much of the trigeminal sensory complex is in a position to supply a monosynaptic drive for lid closure, and the adjacent reticular formation can supply a disynaptic drive. These findings indicate that the assignment of the R1 and R2 components of the blink reflex to different parts of the trigeminal sensory complex cannot be exclusively based on subdivision connectional relationships with facial motoneurons. The characteristics of the R2 component may be due, instead, to other circuit properties.

TRPV1 receptor in the human trigeminal ganglion and spinal nucleus: immunohistochemical localization and comparison with the neuropeptides CGRP and SP.

This work presents new data concerning the immunohistochemical occurrence of the transient receptor potential vanilloid type-1 (TRPV1) receptor in the human trigeminal ganglion (TG) and spinal nucleus of subjects at different ontogenetic stages, from prenatal life to postnatal old age. Comparisons are made with the sensory neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP). TRPV1-like immunoreactive (LI) material was detected by western blot in homogenates of TG and medulla oblongata of subjects at prenatal and adult stages of life. Immunohistochemistry showed that expression of the TRPV1 receptor is mostly restricted to the small- and medium-sized TG neurons and to the caudal subdivision of the spinal trigeminal nucleus (Sp5C). The extent of the TRPV1-LI TG neuronal subpopulation was greater in subjects at early perinatal age than at late perinatal age and in postnatal life. Centrally, the TRPV1 receptor localized to fibre tracts and punctate elements, which were mainly distributed in the spinal tract, lamina I and inner lamina II of the Sp5C, whereas stained cells were rare. The TRPV1 receptor colocalized partially with CGRP and SP in the TG, and was incompletely codistributed with both neuropeptides in the spinal tract and in the superficial laminae of the Sp5C. Substantial differences were noted with respect to the distribution of the TRPV1-LI structures described in the rat Sp5C and with respect to the temporal expression of the receptor during the development of the rat spinal dorsal horn. The distinctive localization of TRPV1-LI material supports the concept of the involvement of TRPV1 receptor in the functional activity of the protopathic compartment of the human trigeminal sensory system, i.e. the processing and neurotransmission of thermal and pain stimuli.

Prolonged Jaw Opening Promotes Nociception and Enhanced Cytokine Expression.

AIMS: To test the hypothesis that prolonged jaw opening, as can occur during routine dental procedures, increases nociceptive sensitivity of the masseter muscle and increases cytokine expression. METHODS: Sprague-Dawley rats were used to investigate behavioral and cellular changes in response to prolonged jaw opening. A surgical retractor was placed around the maxillary and mandibular incisors, and the jaw was held at near maximal opening for 20 minutes. Head-withdrawal responses to mechanical stimuli applied to the facial skin overlying the left and right masseter muscles were determined following jaw opening. Cytokine levels in the upper cervical spinal cord containing the caudal part of the spinal trigeminal nucleus were evaluated using protein antibody microarrays (n = 3). Statistical analysis was performed using a nonparametric Mann-Whitney U test. RESULTS: Prolonged jaw opening significantly increased nocifensive head withdrawal to mechanical stimuli at 2 hours, and days 3 and 7 postinduction (P < .05). The increase in nociceptive response resolved after 14 days. Sustained jaw opening also stimulated differential cytokine expression in the trigeminal ganglion and upper cervical spinal cord that persisted 14 days postprocedure (P < .05). CONCLUSION: These findings provide evidence that near maximal jaw opening can lead to activation and prolonged sensitization of trigeminal neurons that results in nociceptive behavior evoked by stimulation of the masseter muscle, a physiologic event often associated with temporomandibular disorders (TMD). Results from this study may provide a plausible explanation for why some patients develop TMD after routine dental procedures that involve prolonged jaw opening.

Estrogen receptors localization in the spinal trigeminal nucleus: an immunohistochemical study in humans.

There is increasing evidence for estrogenic modulation of neurotransmission within the trigeminal pain pathway. It is also likely that the effects of estrogens may be influenced by the presence and localization of estrogen receptors (ERs) in a given brain area. To date, human data on the localization of ERs in the spinal trigeminal nucleus (STN), a key brain region in craniofacial nociception, are lacking. To ascertain whether ERs are expressed in the human STN, we performed immunohistochemical analysis on medulla oblongata samples taken from eight adult subjects (three men and five women; age range, 23-71 years) who had died from causes unrelated to neurologic or endocrine diseases. Paraffin-embedded sections at the level of the subnucleus caudalis and interpolaris were incubated with anti-estrogen receptor alpha (ERα) and anti-estrogen receptor beta (ERß) antibodies. ERα immunoreactivity was detected in the nucleus and cytoplasm of neuronal and glial cells in the STN and in the nerve fibers within the spinal trigeminal tract in all eight subjects; ERß immunoreactivity was observed in the cytoplasm of neuronal cells in five subjects. This study is the first to provide evidence in humans that ER immunoreactivity is detectable on neuronal and glial cells of the STN. The two ER subtypes exhibited different expression patterns, with higher expression levels of ERα than ERß. The presence of ER-containing cells in the STN suggests that estrogens may directly affect trigeminal neuron excitability in humans.

Extreme tolerance to ammonia fumes in African naked mole-rats: animals that naturally lack neuropeptides from trigeminal chemosensory nerve fibers.

Naked mole-rats (Heterocephalus glaber) naturally lack neuropeptides associated with the signaling of chemical irritants from C type trigeminal nerve fibers. The goal of the present study was to assess behavioral responses of these animals to stimulation of the trigeminal chemosensory system, and to determine if stimulation would increase post-synaptic activity in the trigeminal nucleus, as seen in laboratory mice and rats. The results show that naked mole-rats are behaviorally insensitive to capsaicin solution applied to the nostrils and to ammonia fumes in a behavioral avoidance test. Centrally, the number of c Fos labeled cells in the spinal trigeminal nucleus increased from exposure to ammonia although the magnitude of the increase was less than for rats. The increase observed in naked mole-rats likely reflects activity from glutamate release, which appears insufficient to drive pain and aversion behaviors. The results support the idea that neuropeptides in the C fibers of the trigeminal system may be required to signal the aversive quality of specific chemical irritants. The natural lack of neuropeptides in naked mole-rats may be an adaptation to living in a challenging subterranean environment with extremely high levels of ammonia and carbon dioxide, stimuli known to excite trigeminal chemosensory C fibers.

Organization of diencephalic projections from the spinal trigeminal nucleus oralis: an anterograde tracing study in the rat.

The organization of the efferent projections from the spinal trigeminal nucleus oralis (Sp5O) to the diencephalon was studied in the rat using the anterograde tracer Phaseolus vulgaris leucoagglutinin. The present study confirms the existence of trigemino-thalamic pathways originating from the Sp5O and details their distribution. The main diencephalic targets of the Sp5O are the ventral posteromedial thalamic nucleus (VPM), the posterior thalamic nuclei (Po) and the ventral part of the zona incerta (ZIv), contralaterally, and the parvicellular part of the ventral posterior thalamic nucleus (VPpc), bilaterally. The distribution of these projections varies according to the dorso-ventral location of the injection sites: the dorsal part of the Sp5O projects to the medial part of the VPM and the Po, and to the caudal part of the ZIv, as well as to the VPpc. The ventral part of the Sp5O projects to the lateral part of the VPM and the Po and to the rostral part of the ZIv. These results suggest that the trigemino-diencephalic pathways originating from the Sp5O are involved in the processing of gustatory and somatosensory information.

Organization of parabrachial projections from the spinal trigeminal nucleus oralis: an anterograde tracing study in the rat.

In recent years, we have accumulated data showing that the spinal trigeminal nucleus oralis (Sp5O) contributes to the processing of somatosensory inputs from the orofacial region. Although the parabrachial area (PB) represents the main brainstem relay for autonomic, nociceptive, and gustatory afferents, few data are available regarding the topographical distribution of the efferent projections from the Sp5O to the PB. We have addressed this question with the rat, by using the anterograde tracer Phaseolus vulgaris leucoagglutinin. A dense trigeminoparabrachial pathway from the Sp5O toward, predominantly, the ipsilateral PB was revealed. Projections come mainly from the dorsal part of the Sp5O that was found to innervate densely the medial, external medial, and ventral lateral subnuclei. In contrast, the ventral part of the Sp5O projected almost exclusively to an as yet not formally described region, located dorsally and laterally to the lateral tip of the brachium conjunctivum, close to the Kölliker-Fuse nucleus. These results suggest that distinct regions within the Sp5O may be involved in the processing of gustatory and nociceptive information.

Vagotomy prevents morphine-induced reduction in Fos-like immunoreactivity in trigeminal spinal nucleus produced after TMJ injury in a sex-dependent manner.

Acute injury to the temporomandibular joint (TMJ) region activates neurons in multiple, but spatially discrete, areas of the trigeminal spinal nucleus as seen by an increase in Fos-like immunoreactive neurons (Fos-LI). Pretreatment with morphine greatly reduces Fos-LI produced in the dorsal paratrigeminal area (dPa5), ventrolateral pole of the subnucleus interpolaris/caudalis (Vi/Vc-vl) transition region, and laminae I-II at the subnucleus caudalis/upper cervical cord junction (Vc/C2) suggesting a role for these areas in processing pain signals from the TMJ region. To determine if vagal afferents contribute to neural activation after TMJ injury or reduction of activity after morphine, Fos-LI was quantified in the lower brainstem and upper cervical spinal cord of intact and vagotomized male and female rats under barbiturate anesthesia. Bilateral cervical vagotomy (VgX) did not affect Fos-LI produced by TMJ injury in males or females in the absence of morphine. By contrast, morphine-induced reduction in Fos-LI produced at the Vi/Vc-vl transition region was prevented by prior VgX in males and diestrus females, but not in proestrus females. Morphine inhibition of Fos-LI produced in laminae I-II at the Vc/C2 junction region was diminished in vagotomized males compared to intact animals, but not affected in females. In an autonomic control area, the caudal ventrolateral medulla (CVLM), VgX reversed the morphine-induced reduction in Fos-LI in males and females similarly compared to their respective intact controls. These results were consistent with the hypothesis that the Vi/Vc-vl transition region plays a unique role in deep craniofacial pain processing and may integrate autonomic and opioid-related modulatory signals in a manner dependent on sex hormone status.

Induction of Fos protein-like immunoreactivity in the trigeminal spinal nucleus caudalis and upper cervical cord following noxious and non-noxious mechanical stimulation of the whisker pad of the rat with an inferior alveolar nerve transection.

After transection of the inferior alveolar nerve (IAN: the third branch of the trigeminal nerve), the whisker pad area, which is innervated by the second branch of the trigeminal nerve, showed hypersensitivity to mechanical stimulation. Two days after IAN transection, the threshold intensity for escape behavior to mechanical stimulation of the ipsilateral whisker pad area was less than 1.0 g, a sign of allodynia, and returned to the preoperative level (preoperative threshold: 52.0 g) at 32 days after surgery. This decrement of escape threshold lasted for more than 3 weeks. The whisker pad area contralateral to the IAN transection also showed a decrease in escape threshold to non-noxious mechanical stimulation as compared with sham-operated rats. However, the change in threshold intensity for the side contralateral to transection was not as pronounced as that on the ipsilateral side. Fos protein-like immunoreactive (LI) cells were observed in the superficial laminae but not dominant in deeper laminae of the trigeminal spinal nucleus caudalis (Vc) and the first segment of the spinal cord (C1) after non-noxious mechanical stimulation of the whisker pad area in the rats with IAN transection. Fos protein-LI cells were expressed bilaterally in the Vc and C1, but were more numerous on the ipsilateral side to transection than on the contralateral side. The largest number of Fos protein-LI cells was observed at 2400 microm caudal from the trigeminal subnucleus interporalis (Vi)-Vc border both in ipsilateral and contralateral sides. The number of Fos protein-LI cells increased after application of 1, 4, and 16 g stimuli as compared to rats without mechanical stimulation. Furthermore, an extensively greater number of Fos protein-LI cells were expressed both in superficial and deep laminae of the bilateral Vc and C1 of the spinal cord after subcutaneous injection of mustard oil into the whisker pad. Fos protein expression after mustard oil injection was much stronger than that observed after any mechanical stimulation in the rats with IAN transection. These data suggest that the change in the numbers and spatial arrangement of nociceptive neurons in the Vc and C1 after IAN transection reflect the development of mechanical hyperalgesia in the area adjacent to the IAN innervated region.

Tooth extraction-induced internalization of the substance P receptor in trigeminal nucleus and spinal cord neurons: imaging the neurochemistry of dental pain.

Although pains arising from the craniofacial complex can be severe and debilitating, relatively little is known about the peripheral and central mechanisms that generate and maintain orofacial pain. To better understand the neurons in the trigeminal complex and spinal cord that are activated following nociceptive stimuli to the orofacial complex, we examined substance P (SP) induced internalization of substance P receptors (SPR) in neurons following dental extraction in the rat. Unilateral gingival reflection or surgical extraction of a rat maxillary incisor or molar was performed and tissues harvested at various time points post-extraction. Immunohistochemical analysis of brainstem and cervical spinal cord sections was performed using an anti-SPR antibody and confocal imaging. Both the number and location of neurons showing SPR internalization was dependent on the location and extent of tissue injury. Whereas extraction of the incisor induced internalization of SPR in neurons bilaterally in nucleus caudalis and the spinal cord, extraction of the molar induced strictly unilateral internalization of SPR-expressing neurons in the same brain structures. Minor tissue injury (retraction of the gingiva) activated SPR neurons located in lamina I whereas more extensive and severe tissue injury (incisor or molar extraction) induced extensive SPR internalization in neurons located in both laminae I and III-V. The rostrocaudal extent of the SPR internalization was also correlated with the extent of tissue injury. Thus, following relatively minor tissue injury (gingival reflection) neurons showing SPR internalization were confined to the nucleus caudalis while procedures which cause greater tissue injury (incisor or molar extraction), neurons showing SPR internalization extended from the interpolaris/caudalis transition zone through the C7 spinal level. Defining the population of neurons activated in orofacial pain and whether analgesics modify the activation of these neurons should provide insight into the mechanisms that generate and maintain acute and chronic orofacial pain.

Increased expression of gamma-aminobutyric acid transporters GAT-1 and GAT-3 in the spinal trigeminal nucleus after facial carrageenan injections.

The present study aimed to elucidate the distribution of gamma-aminobutyric acid (GABA) transporters in the spinal trigeminal nucleus after carrageenan injections. Dense GAT-1 and GAT-3 but very little GAT-2 immunoreactivity was observed in the normal rat spinal trigeminal nucleus. The GAT-1-positive glial cells in the normal rat spinal trigeminal nucleus contained dense bundles of glial filaments and had features of astrocytes. Some GAT-3-positive cells contained dense bundles of glial filaments and had features of astrocytes, whilst others lacked glial filaments, and contained dense marginated heterochromatin, and had features of oligodendrocyte precursor cells. An increase in immunoreactivity to both transporters was observed on the injected but not the contralateral side 3 days after facial carrageenan injections. In rats given three further weekly injections of carrageenan and killed 3 days after the fourth injection, further increases in GAT-1 and GAT-3 immunoreactivities were observed. Electron microscopy showed that transporter immunoreactivity in the spinal trigeminal nucleus of carrageenan-injected rats was predominantly present in glial processes, showing that the increase in the number of processes observed at light microscopy was due to increased immunoreactivity in glial processes. An increased expression of GABA transporters in the carrageenan-injected spinal trigeminal nucleus could therefore result in a faster removal of GABA from the synaptic cleft of GABAergic axon terminals compared to normal rats. This could result in reduced inhibition/increased activity of the trigeminothalamic neurons in the spinal trigeminal nucleus, and could contribute to hyperalgesia after carrageenan injections.

c-fos expression in trigeminal spinal nucleus after electrical stimulation of the hypoglossal nerve in the rat.

The expression of the immediate early gene, c-fos, was used to determine the distribution of brainstem neurons activated by stimulation of the distal hypoglossal nerve (XIIn) trunk. The traditional view of the XIIn is one of purely motor function; however, stimulation of XIIn excites neurons in the trigeminal spinal nucleus. The rationale for this study was to use c-fos expression as a marker for postsynaptic activity to define the pattern of brainstem neurons excited by XIIn stimulation. It was further hypothesized that if the afferent fibers that course within XIIn supply deep lingual tissues, then c-fos expression after direct stimulation of XIIn should display a pattern similar to that seen after chemical irritant stimulation of the deep tongue muscle. In barbiturate-anesthetized male rats electrical stimulation of XIIn produced a significant increase in Fos-positive neurons in the dorsal paratrigeminal nucleus (dPa5) and laminae I-II of caudal subnucleus caudalis (Vc) and upper cervical dorsal horn. Mustard oil injection into the deep tongue muscle also produced an increase in c-fos expression in dPa5; however, the highest density of expression occurred in laminae I-II at the dorsomedial aspect of rostral Vc. Both electrical stimulation of XIIn and mustard oil stimulation of the deep tongue increased c-fos expression in the caudal ventrolateral medulla, an autonomic relay nucleus. These results suggest that one site of innervation for afferent fibers that travel within the distal trunk of XIIn is to supply the deep tongue muscle and to terminate in the dPa5. A second group of postsynaptic neurons activated only by XIIn stimulation was located in lamina I-II in caudal portions of Vc and upper cervical dorsal horn, a laminar distribution consistent with a role for XIIn afferents in sensory or autonomic aspects of lingual function.

Nitric oxide synthase and glutamate receptor immunoreactivity in the rat spinal trigeminal neurons expressing Fos protein after formalin injection.

Although recent studies implicated glutamate receptors and nitric oxide in nociception, much still needs to be known about their localisation in neurons involved in nociceptive transmission from the orofacial region. In this study, c-fos expression indicated by Fos immunohistochemistry in the caudal spinal trigeminal nucleus induced by subcutaneous injection of formalin into the lateral face of the rat was used as a marker for nociceptive neurons. The study sought to determine whether Fos-positive neurons express nitric oxide synthase, glutamate N-methyl-D-aspartate type receptor subunit 1, and glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type receptor subunit 2/3; and whether they project to the thalamus. After formalin injection, many Fos-positive nuclei appeared in the superficial laminae of the ipsilateral trigeminal nucleus. Confocal laser scanning microscope revealed that almost all neurons with Fos immunofluorescent nuclei were colocalised with N-methyl-D-aspartate receptor 1, 94% with glutamate receptor 2/3 and 14% with nitric oxide synthase. Some of them were closely related to neurons labelled by nitric oxide synthase. Lastly, some of the Fos-positive neurons were labelled by tetramethylrhodamine-dextran injected into the trigeminothalamic tract or the thalamic region. The results suggested that activation of N-methyl-D-aspartate receptor 1 and glutamate receptor 2/3 upon glutamate release in response to noxious stimulation to the orofacial region might mediate c-fos expression in neurons involved in nociception. The expression of Fos in the neurons could also be mediated by nitric oxide produced from the same, as well as neighbouring neurons, when nociceptive stimulation persisted. Fos-positive neurons in the spinal trigeminal nucleus may project to the thalamus, relaying orofacial nociception to the higher sensory centre.

NMDA receptor subunit NR1-immunoreactivity in the rat pons and brainstem and colocalization with Fos induced by nasal stimulation.

In the present study, we examined the distribution of neurons in the parabrachial nucleus (PB), the Kölliker-Fuse nucleus (KF), the spinal trigeminal nucleus caudalis (Sp5C), the nucleus of the solitary tract (NTS) and the ventrolateral medulla (VLM), which are activated by evoking the nasotrigeminal reflex and which exhibit immunoreactivity for the N-methyl-D-aspartate (NMDA) receptor subunit NR1. By stimulating the nasal mucosa with saline, we induced the expression of the immediate early gene c-fos and combined the immunocytochemical detection of the Fos protein with the detection of the NR1 subunit. Cell counts revealed that nasal stimulation, compared to anesthesia controls, resulted in highly significant increases (p < or = 0.001) of Fos-immunoreactive (-ir) neurons in the midlevel KF, the external lateral PB, and the Sp5C. In the central lateral PB, the rostral ventrolateral medulla including the Bötzinger/pre-Bötzinger complex, and in the ventrolateral and commissural NTS the increases were only moderately significant (p < or = 0.05). With respect to the numbers of NR1-/Fos-ir double-labeled neurons, significant increases were only observed in a subset of these pontomedullary nuclei. Increases were highly significant in the Sp5C (p < or = 0.001) and the midlevel KF (p < or = 0.01) and moderately significant (p < or = 0.05) in the external lateral PB, Bötzinger/pre-Bötzinger complex, and ventrolateral NTS. The present study revealed that nasotrigeminally activated neurons in mandatory and potential relay sites of the nasotrigeminal reflex circuit express the NR1 subunit. This finding strongly suggests that NMDA-type glutamate receptors are involved in the mediation of the nasotrigeminally evoked cardiovascular and respiratory responses.

Substance P receptor (NK1)-immunoreactive neurons projecting to the periaqueductal gray: distribution in the spinal trigeminal nucleus and the spinal cord of the rat.

Substance P receptor (SPR)-immunoreactive neurons projecting to the periaqueductal gray (PAG) were examined in the rat spinal trigeminal nucleus and spinal cord by a retrograde tracing method combined with immunofluorescence histochemistry. After injection of Fluoro-gold (FG) into the PAG, SPR-immunoreactive neurons labeled with FG were observed mainly in the lateral spinal nucleus and lamina I of the medullary and spinal dorsal horns and additionally in laminae V and X of the spinal cord.

The distribution of trigeminovascular afferents in the nonhuman primate brain Macaca nemestrina: a c-fos immunocytochemical study.

An understanding of migraine must be based on data concerning the anatomy and physiology of the painsensitive intracranial structures. Stimulation of the superior sagittal sinus produces changes in brain blood flow and changes in neuropeptide levels similar to those seen in humans during migraine. To better understand the anatomy of the central ramifications of pain-sensitive intracranial structures we have examined the distribution of c-fos immunoreactivity in the monkey when the sinus is stimulated. Six adult Macaca nemestrina monkeys were anaesthetised. The superior sagittal sinus was isolated after a midline craniotomy and a paraffin well created. At 24 h after completion of the surgery the sinus was stimulated electrically for 1 h and the brain subsequently removed and processed for c-fos. In control animals in which the sinus was isolated but not stimulated there was a small amount of c-fos expression in the caudal brainstem and upper cervical spinal cord. Stimulation of the superior sagittal sinus evoked expression of c-fos in the caudal superfical laminae of the trigeminal nucleus and in superficial laminae of the dorsal horn of the C1 level of the upper cervical spinal cord. A lesser amount of c-fos was seen at C2 while no significant labelling above control was observed at C3. These data, while largely confirming the results from the cat concerning the central distribution trigeminovascular afferents, underscore a possibly unique specialisation of trigeminovascular afferents at the C1 level. Given the close evolutionary relationship of the monkey to man it is likely that the cells described in this study represent for primates the nucleus that mediates the pain of migraine.

Distribution of Fos-like immunoreactivity in the caudal brainstem of the rat following noxious chemical stimulation of the temporomandibular joint.

Central expression of the protooncogene c-fos was used to examine areas receiving noxious sensory input from the rat temporomandibular joint (TMJ). Fos-like immunoreactivity (Fos-LI) in the caudal brainstem was visualized 2 hours after unilateral injection of the small-fiber-specific excitant/inflammatory irritant mustard oil into the TMJ region. Control animals received injection of either mustard oil into the subcutaneous fascia overlying the masseter muscle or mineral oil vehicle into the TMJ region. In all groups, Fos-LI was consistently observed ipsilaterally in the spinal trigeminal nucleus and cervical dorsal horn and, bilaterally, in the nucleus of the solitary tract and the ventrolateral medulla. The expression of Fos-LI ipsilaterally in the paratrigeminal nucleus was variable. Within the trigeminal sensory complex, Fos-LI was restricted to subnucleus caudalis and the caudal portions of subnucleus interpolaris near the level of the obex. Approximately 12% of Fos-LI cells in subnucleus caudalis and in the cervical dorsal horn were found in laminae III-VI. Compared to TMJ mustard oil injection, mineral oil injection produced less Fos-LI at all rostrocaudal levels, whereas subcutaneous mustard oil injection produced less Fos-LI in caudal subnucleus caudalis but similar amounts in the cervical dorsal horn. Neither of these injections yielded significant ipsilateral responses in subnucleus caudalis, indicating that Fos-LI in this region following TMJ mustard oil injection could be ascribed solely to small-fiber stimulation in the deep TMJ region. The wide rostrocaudal distribution of Fos-LI within the caudal brainstem reflects the distribution of TMJ-responsive nociceptive neurons that may underlie the spread and referral of pain from the TMJ region.

Production of specific antibodies against GABA transporter subtypes (GAT1, GAT2, GAT3) and their application to immunocytochemistry.

Polyclonal subtype-specific antibodies were developed against three subtypes of GABA transporters (GAT1, GAT2 and GAT3). By immunoblot analysis, each antibody detected a single band that could be blocked by absorption of the antibody with the respective antigen. GAT2 was found in various tissues, while GAT1 and GAT3 were detected only in the brain. GAT1 was distributed throughout the brain with the highest amount in the olfactory bulb, CA3 region of the hippocampus, layer I of the cerebral cortex, piriform cortex, superior colliculus, interpeduncular nucleus and nucleus spinal tract of the trigeminal nerve, while the GAT3 was densely found in the olfactory bulb, thalamus, hypothalamus, pons and medulla, globus pallidus, central gray, substantia nigra, deep cerebellar nuclei and nucleus spinal tract of the trigeminal nerve but not in the hippocampus, cerebral cortex, caudate-putamen and cerebellar cortex. GAT2 immunoreactivity was faint throughout the brain but was concentrated in the arachnoid and ependymal cells. Both GAT1 and GAT3 were found in the neuropil but not in the cell bodies nor in the white matter. These results suggest that GAT1, GAT2 and GAT3 are expressed in different cells and that GAT1 and GAT3 are involved in distinct GABAergic transmission while GAT2 may be related to non-neuronal function.

Two major types of premotoneurons in the feline trigeminal nucleus oralis as demonstrated by intracellular staining with horseradish peroxidase.

Previous studies suggest that neurons in the dorsomedial subdivisions of trigeminal nucleus oralis (Vo) may contribute to reflex control of jaw movements and to modulation of sensory information. The present study has addressed this possibility by the use of intracellular staining with horseradish peroxidase of physiologically identified neurons in Vo to examine functional and morphological properties of these neurons. Of 14 labeled neurons, eight had axon collaterals terminating exclusively in the dorsolateral subdivision of the trigeminal motor nucleus (DL neurons) and four in its ventromedial subdivision (VM neurons); axon collaterals of two neurons were not traced. Both groups of neurons sent terminal arbors into other nuclei of the lower brainstem. The DL neurons were distinguishable from the VM neurons in their receptive field (RF) location, neuronal position, somadendritic architecture, and projections to other brainstem nuclei. All neurons, except for two that were exclusively activated by noxious stimuli applied to the tongue, were responsive to light mechanical stimulation of peri- and intraoral structures. The RFs of the DL neurons were located in more posterior oral structures than those of the VM neurons. The RF of nearly all low-threshold DL neurons was located in the maxillary region, and that of the VM neurons, in contrast, involved the mandibular region. The VM neurons were located medial or ventral to the DL neurons. The soma size of the VM neurons was significantly larger than that of the DL neurons. Dendritic arbors of both groups could be separated into medial and lateral components. The ratio of the dendritic transverse areas in the medial vs. lateral component was significantly higher in the VM neurons than in the DL neurons. The DL neurons also issued collaterals that terminated in larger brainstem areas than those of the VM neurons. These observations provide new evidence on the morphological and functional properties of Vo neurons that contribute to reflex control of jaw and facial movements and modulation of sensory information.

Somatostatin, galanin and peptide histidine isoleucine in the newborn and adult human trigeminal ganglion and spinal nucleus: immunohistochemistry, neuronal morphometry and colocalization with substance P.

By means of indirect immunofluorescence the neuropeptides somatostatin, galanin and peptide histidine isoleucine were localized in cell bodies, nerve fibres and terminal-like elements in the ganglion and spinal nucleus of the human trigeminal nerve in perinatal and adult ages. No immunoreactivity to vasoactive intestinal polypeptide was observed. In the gasserian ganglion somatostatin-, galanin- and peptide histidine isoleucine-containing neurons and nerve fibres occurred frequently in pre- and full-term newborns, but were scarce to absent in adults. Somatostatin- and galanin-positive pericellular basket-like structures around non-immunoreactive perikarya were observed in newborn specimens. Immunoreactivity to somatostatin, galanin and peptide histidine isoleucine labelled nerve fibers and punctate and felt-like nerve terminals in the pars interpolaris and subnucleus caudalis of the spinal trigeminal nucleus, with immunostaining and distribution patterns characteristic for each peptide. In addition, somatostatin-containing neuronal cell bodies frequently were detected. At variance with those containing somatostatin, the number of galanin- and peptide histidine isoleucine-like immunoreactive elements were dramatically reduced in the adult tissue compared to the newborn one. Double immunostaining revealed that each of the three peptides partially colocalizes with substance P, the degree of coexistence being very low for somatostatin/substance P and high for galanin/substance P and peptide histidine isoleucine/substance P both in the gasserian ganglion and in the spinal nucleus. The results obtained suggest that somatostatin, galanin and peptide histidine isoleucine may play functional roles in primary sensory neurons and at the first synaptic level of the human trigeminal sensory system.